Opportunistic fungal pathogens cause serious infections in humans, especially in immunocompromised hosts, such as patients infected with the human immunodeficiency virus (HIV). Even with highly active antiretroviral therapy, opportunistic infections are a considerable problem in patients with advanced HIV disease, in patients who experience failure to treatment, and in patients who discontinue therapy due to adverse drug interactions or economic reasons. These infections are also on the rise in cancer patients and organ transplant recipients due to improved immunosuppressive strategies and intense chemotherapy regimens. Currently available therapies for fungal infections have several limitations including drug resistance, toxicity, drug interactions, high-cost, and severe side effects. New antifungal drugs that act on new molecular targets are greatly needed. The overall goal of this project is to identify new compounds, as well as new pathways, for the development of new treatments for the major AIDS- and cancer-related disseminated fungal infections. This project expands our previous work in the area of antifungal drug discovery, with an emphasis on the mechanistic characterization and validation of antifungal and synergistic compounds. The proposed approach is to determine the mechanism of action of lead compounds, validate the relevance of the mechanisms in fungal pathogens, and discover new lead compounds targeting the new pathways identified. The specific aims of this project are: (1) Determine the mechanism of action of lead antifungal compounds using genomic, genetic and proteomic approaches;(2) Validate the newly identified molecular pathways by determining their relevance in the susceptibility and virulence of fungal pathogens;(3) Identify novel mechanisms of antifungal drug resistance by evaluating new synergistic compounds and new resistance-conferring genes;and, (4) Identify additional new lead compounds with unique mechanistic profiles, including compounds that inhibit newly identified molecular pathways. Successful completion of the proposed work will yield new prototype lead compounds that will serve as templates for development of new classes of antifungal agents, as templates for the development of new drugs to be used in combination with existing anti-fungal agents to reverse resistance to these agents, and as probes to identify novel mechanisms of action that can be exploited in the future search for other new derivatives. This information will expand the foundation of knowledge regarding the therapeutic control of opportunistic fungal infections and the understanding of the basic biology of the pathogens involved. PUBLIC HEALTH RELEVANCE: PROJECT: There is an urgent need for the development of new antifungal drugs for clinical use due to the following reasons: (i) life-threatening fungal infections are a serious public health problem in immunocompromised patients (e.g., patients infected with human immunodeficiency virus, cancer patients, and organ transplant recipients), (ii) current drugs used to treat life-threatening fungal infections are inadequate due to problems related to toxicity, resistance, high cost, serious side effects, and drug interactions, (iii) drug-resistant fungal strains are on the rise, and (iv) new fungal pathogens are continuing to appear. The overall goal of this project is to identify new compounds, as well as new pathways, for the development of new treatments for opportunistic fungal infections. This project will also result in the discovery of new drugs that can be used in combination with existing antifungal agents to improve their activity in resistant pathogens.